Small nuclear ribonucleoprotein particles (snRNPs) catalyze pre-mRNA splicing through a complex series of interactions with the intron and with other snRNPs. While some of the roles of the snRNAs in splicing have been discovered, the functions of their associated proteins remain almost entirely unknown. Recent evidence suggests that the protein components of the U1 snRNP play essential roles in early spliceosome formation. The 70K protein is a component of the U1 snRNP and is required for efficient splicing. We have shown that the amino terminal 100 amino acids of 70K are necessary and sufficient for its function. Our initial goal is to identify precisely which residues of the amino terminus are essential. We will create random mutations in the amino terminus of 70K and test their ability to provide 70K function. During splicing, the U1 snRNP interacts with the 5' splice site, the branch site, and a series of other splicing factors to form a "bridge" between the ends of the intron. Experiments in mammalian cell extracts indicate that 70K is likely to play a pivotal role in these interactions. Therefore, we seek to identify yeast factors that interact with 70K, as this will be essential for understanding how snRNPs recognize and juxtapose splice sites. To do so, we will employ a set of three genetic techniques; suppressor screening, synthetic lethal screening, and two hybrid screening. These techniques will be applied to specific yeast target proteins that are known or postulated to interact with 70K. In addition, we will use the same three techniques in an unbiased test to identify gene products that interact with 70K. Interactions between 70K and gene products identified will be verified using biochemical techniques. We will also analyze PRP4O, an essential yeast gene and is required for splicing in vivo and in vitro. These splicing phenotypes and its physical association with the U1 snRNP strongly suggest that Prp40 plays an important part in U1 snRNP function. The experiments described in this proposal are designed to elucidate the interactions formed by two yeast U1 snRNP proteins. Because the U1 snRNP performs a crucial role in early spliceosome formation, this information will directly help us to understand how the splice sites are identified within the pre-mRNA, and how they are brought into juxtaposition for splicing.